Spheroids of FAP-Positive Cell Lines as a Model for Screening Drugs That Affect FAP Expression

Abstract

Fibroblast activation protein has a unique expression profile that manifests mainly in wounds and tumors, which anticipates it as an encouraging and selective target for anticancer therapy. However, research of the therapeutic potential of FAP is limited both by legal restraints when working in vivo and by the difficulty of obtaining standardized primary cultures of FAP-positive cancer-associated fibroblasts due to their high heterogeneity. We found that 3D spheroids of FAP-positive cell lines could serve as robust and convenient models of FAP expression, in contrast to monolayers. By exposing such spheroids to various factors and compounds, it is possible to study changes in FAP expression, which are easily detected by confocal microscopy. FAP expression increases under the influence of the TGFβ, does not depend on pH, and decreases during hypoxia and starvation. We believe that the proposed model could be used to organize large-scale high-throughput screening of drugs that target FAP expression.

Keywords: 3D spheroids; cancer-associated fibroblasts; fibroblast activation protein.

Figure 1

Representative confocal microscopy images of FAP-positive cell line spheroids. FAP expression (green) is co-localized with cell membranes (red). The name of the cell line is indicated on the left side. The RMS cell line is shown at a higher magnification than the OSA line. The scale (white horizontal line) is indicated in the lower right corner and corresponds to 10 µm.

Figure 2

Z-stack for spheroid of the RMS cell line. In the upper left corner, there is an image corresponding to the most extreme position of the start of scanning the spheroid. From left to right and further down, the scan layer shifts. It can be seen that the highest concentration of the FAP protein is located exactly on the surface of the spheroid.

Figure 3

FAP expression is increased by adding TGFβ to OSA spheroids. Upper side—relative transcription levels of the FAP gene when a spheroid is cultured in DMEM/F12 containing 0.5% FCS with or without supplementation of the TGFβ. The transcription level was calculated relative to the geometric mean level of transcription of the 18S, GPI, EEF1A1 genes. Lower side—representative confocal microscopy images of a spheroid grown in a DMEM/F12 medium containing 0.5% FCS with or without supplementation of the TGFβ.

Figure 4

An increase in the level of FAP expression in spheroids of cell lines OSA, RMS 13, NGP-127 (cell line names are indicated at the top of the figure) during cultivation without changing the medium. Cell spheroids were cultured in DMEM/F12 medium for 72 h with a single medium change (upper row of the images) or no medium change (lower row of the images).

Figure 5

FAP expression upon incubation of OSA spheroids under different conditions. Conditions for cultivation and passage of cells used for spheroid formation are indicated above the confocal image of the spheroids. The transcription level of FAP gene was calculated relative to the geometric mean level of transcription of the 18S, GPI, EEF1A1 genes.